Abstract:

In various aspects, the present teachings provide systems and methods for
reducing chemical noise in a mass spectrometry instrument that use a
neutral chemical reagent and one or more mass filters to reduce
interfering chemical background ion signals that are generated by
ionization sources of mass spectrometers. In various embodiments, the
neutral chemical reagent belongs to the class of organic chemical species
containing a disulfide functionality.

Claims:

1-36. (canceled)

37. A mass spectrometer comprising:a. an ion source that generates ions of
an analyte of interest;b. a mass separator that receives ions generated
by the ion source, the mass separator substantially separating ions in a
first range of mass-to-charge ratio values and transmitting at least a
portion of the ions in the first range of mass-to-charge ratio values
through an exit;c. a reaction region containing a reactive gas, the
reaction region receiving the ions transmitted through the exit of the
mass separator and causing collisions of at least a portion of the
transmitted ions with the reactive gas that reacts with one or more ionic
species, but that does not substantially react with the ions of the
analyte of interest, thereby allowing only ions with mass-to-charge ratio
values in a second mass-to-charge ratio range to be extracted from an
exit of the reaction region; andd. an ion detector that detects ions
extracted from the reaction region, the ion detector detecting the ions
of the analyte of interest with a level of noise that is reduced by the
collisions in the reactive region, wherein a mass-to-charge ratio of the
ions of the analyte of interest can be determined from the ion detection.

41. The mass spectrometer of claim 40 wherein the neutral chemical agent
comprises an organic chemical species containing a disulfide.

42. The mass spectrometer of claim 37 further comprising a second mass
separator that receives ions extracted from the exit of the reaction
region, the second mass separator substantially separating ions in a
third mass-to-charge ratio range and transmitting at least a portion of
the ions in the third mass-to-charge ratio range through an exit to the
ion detector.

43. The mass spectrometer of claim 37 further comprising an ion mobility
spectrometer that receives ions extracted from the reaction region, the
ion mobility spectrometer substantially separating ions in a range of ion
mobility values that corresponds to a third mass-to-charge ratio range
and transmitting the separated ions through an exit to the ion detector.

44. The mass spectrometer of claim 37 further comprising a bandpass mass
filter positioned proximate to the exit of the reaction region, the
bandpass mass filter passing only ions with mass-to-charge ratios that
are within a predetermined mass-to-charge ratio range to the ion
detector.

46. The mass spectrometer of claim 37 further comprising a high-pass mass
filter positioned proximate to the exit of the mass separator before the
reaction region, the high-pass mass filter passing only ions with
mass-to-charge ratios that have masses greater than a predetermined
mass-to-charge ratio value to the reaction region.

47. The mass spectrometer of claim 37 further comprising a gas flow
controller that adjusts a partial pressure of the neutral chemical
reagent to change an efficiency of ion-molecule reactions in the reaction
chamber.

48. A mass spectrometer comprising:a. an ion source that generates ions of
an analyte of interest;b. an ion mobility spectrometer that receives ions
generated by the ion source, the ion mobility spectrometer substantially
separating ions in a range of ion mobility values and transmitting at
least a portion of the ions in the range of ion mobility values through
an exit;c. a reaction region containing a reactive gas, the reaction
region receiving the ions transmitted through the exit of the ion
mobility spectrometer and causing collisions of at least a portion of the
transmitted ions with the reactive gas that reacts with one or more ionic
species, but that does not substantially react with the ions of the
analyte of interest, thereby allowing only ions with mass-to-charge ratio
values in a first selected mass-to-charge ratio range to be extracted
from an exit of the reaction region; andd. an ion detector that detects
ions extracted from the reaction region with a level of noise that is
reduced by the collisions in the reactive region, wherein a
mass-to-charge ratio of the ions of the analyte of interest can be
determined from the ion detection.

49. The mass spectrometer of claim 48 wherein the ion mobility
spectrometer separates ions in the first range of ion mobility values by
both steady-state ion mobility and by differential ion mobility.

50. The mass spectrometer of claim 48 wherein the range of ion mobility
values is a range of ion mobility values that correspond to a range of
mass-to-charge ratios that is below a selected range of mass-to-charge
ratio values.

52. The mass spectrometer of claim 51 wherein the neutral chemical agent
comprises an organic chemical species comprising a disulfide.

53. The mass spectrometer of claim 48 further comprising a second ion
mobility spectrometer that receives ions extracted from the exit of the
reaction region, the second ion mobility spectrometer substantially
separating ions in a second range of ion mobility values and transmitting
at least a portion of the ions in the second range of ion mobility values
through an exit to the ion detector.

54. The mass spectrometer of claim 48 further comprising a mass separator
that receives ions extracted from the exit of the reaction region, the
mass separator substantially separating ions in a second range of
mass-to-charge ratio range and transmitting at least a portion of the
ions in the second mass-to-charge ratio range through an exit to the ion
detector.

55. The mass spectrometer of claim 48 further comprising a bandpass filter
positioned proximate to the exit of the reaction region, the bandpass
filter passing only ions with mass-to-charge ratios that are within a
predetermined mass-to-charge ratio range.

57. The mass spectrometer of claim 48 further comprising a high-pass mass
filter positioned proximate to the exit of the ion mobility spectrometer
before the reaction region, the high-pass mass filter passing only ions
with mass-to-charge ratios greater than a predetermined mass-to-charge
ratio value to the reaction region.

58. The mass spectrometer of claim 48 further comprising a gas flow
controller that adjusts a partial pressure of the neutral chemical
reagent to change an efficiency of ion-molecule reactions in the reaction
chamber.

59. A mass spectrometer comprising:a. an ion source that generates ions of
an analyte of interest;b. a first mass separator that receives ions
generated by the ion source, the first mass separator substantially
separating ions below a selected mass-to-charge ratio value and
transmitting at least a portion of the ions with mass-to-charge ratio
values above the selected mass-to-charge ratio through an exit;c. a
reaction region containing a reactive gas, the reaction region receiving
the ions transmitted through the exit of the mass separator and causing
collisions of at least a portion of the transmitted ions with the
reactive gas that reacts with one or more ionic species, but that does
not substantially react with the ions of the analyte of interest, thereby
allowing only ions with mass-to-charge ratio values in a first selected
mass-to-charge ratio range to be extracted from an exit of the reaction
region;d. a second mass separator that receives ions exiting the reaction
region, the second mass separator substantially separating ions in a
second selected mass-to-charge ratio range and transmitting at least a
portion of the ions in the second selected mass-to-charge ratio range
through an exit; ande. an ion detector that detects ions extracted from
the second mass separator, the ion detector detecting the ions of the
analyte of interest with a level of noise that is reduced by the
collisions in the reactive region, wherein a mass-to-charge ratio of the
ions of the analyte of interest can be determined from the ion detection.

60. The mass spectrometer of claim 59 wherein at least one of the first
and the second mass separator comprise a quadrupole mass spectrometer.

61. A mass spectrometer comprising:a. a means for generating ions of an
analyte of interest;b. a means for separating ions generated by the ion
source that have mass-to-charge ratio values in a first selected range;c.
a means for reacting ions in the first selected range of mass-to-charge
ratio values to causes collisions of at least a portion of the selected
ions with a reactive gas that reacts with one or more ionic species, but
that does not substantially react with the ions of the analyte of
interest, thereby allowing only ions with mass-to-charge ratio values in
a second selected mass-to-charge ratio range to be extracted from an exit
of the reaction region; andd. a means for detecting ions of the analyte
of interest with a level of noise that is reduced by the collisions in
the reactive region, wherein a mass-to-charge ratio of the ions of the
analyte of interest can be determined from the ion detection.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]The present application claims the benefit of and priority to
copending U.S. provisional application No. 60/765,809 filed Feb. 7, 2006,
the entire contents of which are herein incorporated by reference.

INTRODUCTION

[0002]The interference of background ions (chemical noise) has been a
problem since the inception of mass spectrometry. This is most acute when
analytes with a low concentration, low ionization efficiency, or both,
are studied. Chemical noise can arise in a variety of mass spectrometry
ion sources such as, for example, an electrospray ionization (ESI),
matrix-assisted laser desorption ionization (MALDI), atmospheric pressure
chemical ionization (APCI), and atmospheric pressure photoionization
(APPI) sources. For example, ESI ion sources can serve as a means for
introducing an ionized sample that originates from a LC column into a
mass separator apparatus. Attempts have been made to reduce chemical
noise in HPLC-MS using either hardware or software approaches, however,
chemical noise can remain even when an improved interface for
de-clustering and high purity HPLC solvents are used.

[0003]MALDI spectra, in particular in the low mass region of the spectra
where small molecule molecular ions reside, are often dominated by
chemical noise to a much greater extent than ESI spectra. It is believed
that the majority of this chemical noise is due to matrix molecules. The
problem can be so great as to preclude the use of systems using MALDI ion
sources from qualitative small molecule analytical applications. Over the
past several years, the scientific community has directed great effort at
solving this problem by attempting to develop matrixless MALDI surfaces.
However, the matrixless approach can result in both a loss of sensitivity
and lead to irreproducibility compared to conventional matrix systems
which transfer the laser energy via the matrix to ionize analytes.

SUMMARY

[0004]This present teachings provide various methods that use a neutral
chemical reagent and one or more mass filters to reduce interfering
chemical background ion signals that are generated by ionization sources
of mass spectrometers. In various embodiments, the neutral chemical
reagent belongs to the class of organic chemical species containing a
disulfide functionality.

[0005]In various aspects, the present teachings present a novel mass
spectrometric approach to reduce the chemical interference in LC-MS,
which can be realized by reactions between chemical background ions and a
chemical reagent combined with an arrangement of band-pass filters based
on ion mobility, mass-to-charge ratio, or both, e.g., an arrangement
using a mass scanning/filtering function of quadrupoles. This technique
has been implemented on a standard triple quadrupole LC-MS, and can be
optimized on a dedicated LC-MS instrumentation.

[0006]We have discovered that a chosen chemical reagent, such as dimethyl
disulfide and ethylene oxide, etc., react substantially exclusively with
the major chemical background ions rather than with the protonated
analytes (for example, small molecule pharmaceuticals and peptides) in
LC/MS. It is believed, without being held to theory, that this is most
likely due to the difference in structures between most chemical
background ions and conventional protonated molecules. Chemical
background ions are mainly classified as either cluster-related ions or
stable ions of (degraded) contaminants (airborne or from the tubing and
solvents).

[0007]The reactions are efficient and can fit well with the pressure
encountered in the ion source, mass analyzer, or both, and can match the
scan speed of a quadrupole MS. While combined with the zero neutral loss
scan mode of a triple quadrupole LC-MS, the exclusive reactions can be
applied, for example, to selectively reduce the level of chemical
background noise and improve the signal-to-noise ratio in the LC/MS of
organic analytes. The present teachings present examples of tests on a
variety of types of analyte ions, which indicate a generic and practical
application of the techniques of the present teachings. In various
embodiments, a reduction of baseline noise in LC/MS by a factor of 10-30
and an improvement of signal-to-noise ratio 5-10 times can be achieved.
The noise reduction thus afforded could be useful for both quantitative
and qualitative analyses, small molecule applications of all types as
well as large molecule proteomic applications.

[0008]The chemical noise reduction methods of the present teachings can be
used with a variety of mass spectrometry and ion mobility systems and
analytical techniques. Mass spectrometry systems to which various
embodiments of the present teachings can be applied include, but are not
limited to, those comprising two mass separators with a collision cell
disposed in the ion flight path between the two mass separators, those
comprising two ion mobility mass separators with a collision cell
disposed in the ion flight path between them; and combinations of a mass
separator and an ion mobility separator with a collision cell disposed in
the ion flight path between them. In various embodiments, a single mass
separator or ion mobility separator can be used where reactions with the
chemical reagent are confined towards the exit portion of the separator.

[0009]Examples of suitable mass separators include, but are not limited
to, quadrupoles, RF multipoles, ion traps, time-of-flight (TOF), and TOF
in conjunction with a timed ion selector. Examples of suitable ion
mobility separators include, but are not limited to, differential ion
mobility spectrometers analyzers (DMS) also referred to as high field
asymmetric waveform ion mobility spectrometers (FAIMS), and substantially
symmetric field ion mobility spectrometers (IMS), all of which can be
used in conjunction with a timed ion selector to provide, e.g., an ion
filtering function. The present teachings can be applied, in various
embodiments, to reduce chemical noise originating from a variety of ion
sources including, but not limited to, an electrospray ionization (ESI),
matrix-assisted laser desorption ionization (MALDI), surface-enhanced
laser desorption ionization (SELDI), atmospheric pressure chemical
ionization (APCI), and atmospheric pressure photoionization (APPI)
sources.

[0010]Examples of mass spectrometry systems to which various embodiments
of the present teachings can be applied include, but are not limited to,
those which comprise one or more of a triple quadrupole, a
quadrupole-linear ion trap (e.g., 4000 Q TRAP® LC/MS/MS System, Q
TRAP® LC/MS/MS System), an LC/MS/MS system (API 5000®, API
4000®, API 3000®, API 2000®, etc.), a quadrupole TOF (e.g.,
QSTAR® LC/MS/MS System), and a TOF-TOF. Examples of mass spectrometry
analytical techniques to which various embodiments of the present
teachings can be applied include, but are not limited to, various forms
of parent-daughter ion transition monitoring (PDITM) such as, for
example, what are referred to as selective ion monitoring (SIM) and
multiple reaction monitoring (MRM) techniques.

[0011]In various embodiments of the teachings described herein, the
neutral chemical reagent can be applied to substantially selectively
reduce the level of chemical background noise and improve the
signal-to-noise ratio in mass spectrometry of organic analytes. In
various embodiments, this approach can be implemented on a triple
quadrupole mass spectrometer by addition of the chemical reagent to the
collision cell and operating the mass spectrometer in the zero neutral
loss scan mode. Various embodiments of such operation are illustrated
schematically in FIG. 1. In various embodiments, implementation of this
noise reduction method can be achieved by adding the chemical reagent to
a reaction region where an arrangement of a low mass filter prior to the
reaction region (e.g., a filter that excludes ions below a selected
mass-to-charge ratio value (m/z) from entering the reaction region), and
a low and high mass filter after the reaction cell (e.g., a band pass
filter that passes ions with an m/z value in a selected range of m/z
values). In various embodiments, this approach can be implemented on a
ion mobility based spectrometer, e.g., comprising two ion mobility
separators (e.g., an DMS and IMS, two IMS, two DMS, etc.) with a
collision cell between them.

[0012]Various embodiments of such arrangements, for example, use of a
bandpass mass filter after the reaction cell in the optics region of the
vacuum chamber prior to the mass analyzer, are illustrated schematically
in FIGS. 2A-2C and 3A-3C. In various embodiments, such filters could be
constructed from one or more high-field assymetric waveform ion mobility
spectrometry (FAIMS) devices located in the atmospheric ion source
region, see, for example, FIG. 3c. The flexibility of such an arrangement
can provide, for example, a triple quadrupole instrument to benefit from
a chemical noise reduction method of the present teachings when operating
in all scan modes. In various embodiments, can also provide for
implementation of the present teachings on other types of mass
spectrometers including, but limited to, TOF, linear and 3-D traps,
Fourier transform mass spectrometers (FTMS), orbit traps, and magnetic
sector instrumentations. For example, in various embodiments, the use of
a chemical reagent and a band pass mass filter prior to the mass
analyzer, could be used as a means to reduce the space charge effects on
ion trapping type mass analyzers as well as to reduce chemical noise in
these instrumentations.

[0013]In various embodiments, the reduction of chemical noise facilitated
by the present teachings can be useful for both quantitative and
qualitative analyses, small molecule applications of all types as well as
large molecule proteomic applications.

[0014]Various embodiments of the present teachings can facilitate
improving signal/noise in both quantitative and qualitative applications
of mass spectrometry. In various embodiments, the present teaching can be
used in combination with other techniques for chemical noise reduction.
For example, because the present teachings can reduce chemical noise
before detection occurs, in various situations the present teachings can
provide additive improvements to software methods such as, e.g., dynamic
background subtraction, and other data processing methods currently in
use. In various embodiments, the present teachings can be used in
situations where LC is not used as a means of sample introduction (e.g.,
nanoESI infusion type methods) where, for example, background subtraction
methods do not work because there are no analyte free regions in the data
from which to derive a background spectra.

[0015]In various aspects, the present teachings provide articles of
manufacture where the functionality of a method of the present teachings
is embedded as computer-readable instructions on a computer-readable
medium, such as, but not limited to, a floppy disk, a hard disk, an
optical disk, a magnetic tape, a PROM, an EPROM, CD-ROM, or DVD-ROM.

[0016]The forgoing and other aspects, embodiments, and features of the
teachings can be more fully understood from the following description in
conjunction with the accompanying drawings. In the drawings like
reference characters generally refer to like features and structural
elements throughout the various figures. The drawings are not necessarily
to scale, emphasis instead being placed upon illustrating the principles
of the teachings.

[0018]FIGS. 2A-C schematically depict various embodiments of band pass
filter arrangements prior to the mass analyzer. Where FIG. 2A
schematically depicts a low resolution quadrupole based filters that can
simulate a zero neutral loss experiment in the Q0 region of the mass
spectrometer of FIG. 1, and having separate high pressure cells for
pre-reaction filtering (high pass filter), reaction, and post-reaction
filtering (band pass filtering); where FIG. 2B schematically depicts an
arrangement similar to FIG. 2A but combining the post-reaction filter and
the reaction cell; and where FIG. 2C schematically depicts an arrangement
where Q0 serves as pre-reaction high pass filter, the reaction cell is in
millitorr Q0 region, and Q1 serves as post-reaction band pass filter.

[0019]FIGS. 3A-C schematically depict various embodiments of band pass
filter arrangements prior to the mass analyzer. Where FIG. 3A
schematically depicts Q0 serving as a pre-reaction high pass filter and
reaction gas (neutral chemical reagent) is added to the entrance of Q1
where reactions and post-reaction filtering occurs; where FIG. 3B
schematically depicts an arrangement where Q1 serves as both pre and
post-reaction filter and reaction gas (neutral chemical reagent) is added
to the middle of the quadrupole in a fashion where reactions do not
substantially occur in the front, high pass filter region; and where FIG.
3C schematically depicts an arrangement where ion mobility filters are in
the atmospheric ion source region based on FAIMS mobility and with
addition of the chemical reagent gas to the drift gas in the middle of a
FAIMS cell wherein the front portion of the reaction cell would filter
pre-reaction and the back half of the reaction cell would filter
post-reaction. It is to be understood that the FAIMS cell can comprise
multiple FAIMS regions with reaction gas added to one or more of these
regions. Multiple FIAMS cells can facilitate, for example, the use of one
or more different drift gases, drift voltages, and combinations thereof.

[0020]FIGS. 4A-4B depict examples of ESI background reduction when using
DMDS in the collision cell in zero neutral loss (ZNL) mode is compared to
using nitrogen but no DMDS in the collision cell. FIG. 4A depicting mass
spectra without DMDS reaction gas and FIG. 4B mass spectra with the
addition of DMDS to the collision cell. The reactions occur with an
estimated 95% of the total chemical background ions from this LC/MS
mobile phase and others tested with electrospray ionization.

[0021]FIG. 5 depicts the effect on the total ion current (TIC) when DMDS
is applied and ZNL scanning where regions correspond to the following:
(a) DMDS added to cell; (b) no gas added to cell; and (c) only nitrogen
added to the cell.

[0022]FIGS. 6A and 6B depict, respectively, mass spectra under the
conditions of regions (a) and (c) of FIG. 5.

[0032]FIGS. 16A-16D compare Angiotensin II background reduction with DMDS.
FIGS. 16A (a) and 16B (b) compare a Q3 single MS scan (with N2 in the
collision cell) with a zero neutral loss with nitrogen. This comparison
shows that the ion current is reduced by about 2.5-3× by virtue of
transmission losses to be expected when operating two RF/DC quadrupoles
instead of one. FIG. 16C (c) and 16(d) compare the effect of DMDS at two
different pressures.

[0033]FIG. 17 depicts a product ion scan of the [M+2H]2+ of
Angiotensin II with DMDS in the cell at a 2 eV collision energy.

[0034]FIGS. 18-20 are tables summarizing the extent of reaction of DMDS
with a variety of compounds in the examples.

[0035]FIGS. 21A-21C assess the reactions of the background ion m/z 99 at
different partial pressures of DMDS using product ion scanning above and
below the mass of the targeted ion. Clusters of water and DMDS are
observed. This m/z=99 ion was determined to be P(OH)4.sup.+ and is
schematically illustrated, e.g., in FIG. 26.

[0036]FIGS. 22A-22D assess the reactions of four background ions as
indicated in the figure header, m/z=83, m/z=115, m/z=143, and m/z=159,
respectively.

[0037]FIGS. 23A-F assess the reactions of an additional six background
ions which do not show extensive adduction but proceed by charge
transfer. The spectra of FIGS. 23A-F are, respectively, the product ion
scans of (a) m/z=149; (b) m/z=60; (c) m/z=78; (d) m/z=83; (e) m/z=99; and
(f) m/z=205.

[0038]FIGS. 24A-B schematically depicts a summary of the reactivity and
believed reaction channels of the background ions in a typical ESI
spectrum of the examples with DMDS. A few of the background ions showed
substantially no reactivity (circled ions).

[0039]FIGS. 25 and 26 schematically summarize a study undertaken to
identify common background ions using various MS/MS scan modes to
establish the relationships among the ion populations.

[0043]FIGS. 30-38 depict chemical structures of various compounds listed
in the tables of FIGS. 18-20.

DESCRIPTION OF VARIOUS EMBODIMENTS

[0044]In various aspects, the present teachings provide systems and
methods for reducing chemical noise in a mass spectrometry instrument. In
various embodiments, the methods comprise: (a) substantially excluding
ions below a selected mass-to-charge ratio value (m/z) from entering a
reaction region and transmitting at least a portion of the ions with a
m/z value above the selected m/z value to the reaction region; (b)
colliding at least a portion of the transmitted ions with a neutral
chemical reagent in the reaction region; and (c) extracting from the
reaction region at least a portion of ions with a m/z value in a selected
m/z range and substantially excluding from extraction ions with a m/z
value outside the selected m/z range; wherein the neutral chemical
reagent reacts with one or more ionic species in the reaction region but
does not substantially react with one or more analytes of interest
transmitted to the reaction region. It is to be understood that as added
into a reaction region, the neutral chemical reagent is also referred to
herein as the reactive gas.

[0045]In various embodiments, the methods comprise: (a) substantially
excluding ions in a selected range of ion mobility values from entering a
reaction region while transmitting at least a portion of ions from the
ion source with an ion mobility value outside the selected range of ion
mobility values; (b) colliding at least a portion of the transmitted ions
with a neutral chemical reagent in the reaction region; and (c)
extracting from the reaction region at least a portion of ions with a m/z
value in a selected m/z range and substantially excluding from extraction
ions with a m/z value outside the selected m/z range; wherein the neutral
chemical reagent reacts with one or more ionic species in the reaction
region but does not substantially react with one or more analytes of
interest transmitted to the reaction region. It is to be understood as
used herein that term ion mobility, includes both steady-state ion
mobility and differential ion mobility. The steady-state ion mobility can
be represented by the equation v=KE, where v is the steady-state ion
drift velocity, K is the steady-state ion mobility, also referred to as
scalar ion mobility, and E is the electrical field intensity.

[0046]In the present teachings, a reaction product is preferably formed
between the neutral chemical reagent and one or more background ion
species, to cause the mass-to-charge ratio's of a background ion to shift
to a higher or lower m/z value than the mass of the original background
ion. The partial pressure of the neutral chemical reagent can be adjusted
such that the ion-molecule reactions are efficient enough so that the
reaction region can be coupled to the spectrometry system scan speed. In
various embodiments, the present teachings combine the use of the neutral
chemical reagent with the scanning and mass filtering properties of a
triple quadrupole operating in the zero neutral loss (ZNL) mode, such
that chemical noise ions (background ions) below the mass of the analyte,
above the mass of the analyte, or above and below the mass of the
analyte, are substantially ejected before reaching the reaction region
(e.g., collision cell) and thus not allowed to react up into the mass
channel of the analyte of interest. Chemical noise ions (background ions)
isobaric with the analyte interest that react with the neutral chemical
reagent gas, move to a higher or lower m/z values and can then be
rejected by a mass filter (e.g. quadrupole, ion selector) situated
between the reaction region and the detector of the mass spectrometry
system. In various embodiments, by applying this concept with a low
resolution band-pass mass or mobility filters prior to the detector, this
noise reduction technique can be applied to all scan modes of a triple
quadrupole by linking the scan of the filter to the scan of the first
quadrupole analyzer. Collecting the chemical noise purified ion
population exiting the filters in a trap can be used, for example, in
various embodiments to extend the technique to all mass analyzer systems.

[0047]In various embodiments, the methods comprise: (a) substantially
excluding ions in a first selected range of ion mobility values from
entering a reaction region while transmitting at least a portion of ions
from the ion source with an ion mobility value outside the first selected
range of ion mobility values; (b) colliding at least a portion of the
transmitted ions with a neutral chemical reagent in the reaction region;
and (c) extracting from the reaction region at least a portion of ions
with an ion mobility value in a second selected ion mobility range and
substantially excluding from extraction ions with an ion mobility value
outside the second selected ion mobility range; wherein the neutral
chemical reagent reacts with one or more ionic species in the reaction
region but does not substantially react with one or more analytes of
interest transmitted to the reaction region. In various embodiments, a
reaction product is formed between the neutral chemical reagent and one
or more background ion species, to cause the ion mobility of a background
ion to shift to a higher or lower ion mobility value than that of the
original background ion.

[0048]In various embodiments the analytes of interest are organic
molecules such as, for example, proteins, peptides and small molecule
pharmaceuticals. In various embodiments, the analytes of interest
comprise cysteine containing peptides.

[0049]In various embodiments where the background ions to be reduced or
removed are positive ions, the neutral chemical reagent is preferably a
nucleophile. In various embodiments where the background ions to be
reduced or removed are negative ions, the neutral chemical reagent is
preferably an electrophile. For example, suitable electrophiles include a
molecules that have an electron withdrawing group that can attach itself
to localized negative charges.

[0050]In various embodiments, the neutral chemical reagent is provided in
the reaction region at an absolute pressure in the range between about
1×10-4 torr and about 760 torr. In various embodiments, the
neutral chemical reagent is provided in the reaction region at an
absolute pressure in the range between: (a) about 5×10-4 torr
and about 8×10-3 torr; (b) about 1×10-3 torr and
about 10×10-3 torr; and/or (c) about 1×10-4 torr
and about 6×10-3 torr.

[0051]In various embodiments, the neutral chemical reagent comprises an
organic chemical species containing a disulfide functionality. Examples
of disulfides include, but are not limited to, dimethyl disulfide and
diethyl disulfide. In various embodiments, the neutral chemical reagent
comprises an organic chemical species containing a diselenide
functionality. An example of a diselenide includes, but is not limited
to, dimethyl diselenide, (CH3Se--SeCH3); it should be noted
that this compound is considered highly toxic. In various embodiments,
the neutral chemical reagent comprises ethylene oxide.

[0052]In various embodiments, the neutral chemical reagent is dimethyl
disulfide (DMDS) (CH3--S--S--CH3; DMDS; CAS no.: 624-92-0;
formula: C2H6S2). In various embodiments of the present teachings, it has
been found that when added to a collision cell, DMDS reacts with
background ions that tend to be composed of clusters yet does not
substantially react with many organic analytes of interest. It has been
observed that the reaction of DMDS with background ions can shift the
mass of the background ion (1) up by the mass of DMDS or several DMDS
molecules; (2) up by the mass of a fragment of DMDS; and/or (3) down by a
charge exchange process and abstraction of a portion of the background
ion. As a result, once a reaction product is formed between the DMDS and
a background ion species, the m/z value of the background ion will shift
to higher or lower value than the mass of the original ion. Accordingly,
it has been discovered that in combination with the use of the neutral
chemical reagent in the reaction region, the use of a high pass mass
filter before the reaction region, and a low resolution high and low mass
filter (band pass filter) after reaction region can be used to remove the
background ions yet leave analyte ions of interest largely undiminished.
As discussed further below, the smallest mass shift observed in the
examples presented herein using DMDS as a neutral chemical reagent was
the production of m/z 141 from m/z 149. The etiology of this ion can be
further understood by reference to FIG. 23A and accompanying text.
Accordingly, in various embodiments, the width of the post-reaction mass
filter is no greater than about ±8 amu.

[0053]In the present teachings, the selection of the neutral chemical
reagent can be based on the chemical reactivity differences between
analyte ions and chemical background ions when they react with the
neutral reagent in the gas phase. It is believed, without being held to
theory, that chemical background (noise) ions can be classified mainly as
either cluster-related ions (e.g., due to insufficient de-clustering or
re-clustering, etc.) or stable ions and their fragments of contaminants
(e.g., airborne or from tubing and solvents, etc.). In LC/MC systems, for
example, cluster-type ions are often HPLC solvent/buffer-related species.

[0054]In various embodiments, the reaction region comprises a collision
cell. Examples of various collision cell arrangements include, but are
not limited to, those illustrated in FIGS. 2A-2C. In various embodiments,
the reaction region is at least partially within a mass separator or ion
mobility separator of the instrument. Examples of such reaction region
arrangements include, but are not limited to, those illustrated in FIGS.
3A-3C.

[0055]In various embodiments, the sample is doped with one or more of
ammonium, an alkali ion (such as, e.g., sodium), or a combination
thereof, to provide adduct ions of the background species. In various
embodiments of a chemical reagent, it was observed that adducted
background ions (e.g., sodiated background ions, background ions adducted
with ammonium, etc.) reacted with DMDS as a chemical reagent to a greater
degree than adduct free background ions. In various embodiments, one or
more of ammonium, alkali ion, or a combination thereof, are doped into
the sample solution prior to ionization in the range between about 0.1
millimolar to about 10 millimolar.

[0056]In various aspects of the present teachings, the post-reaction
region mass filter can be scanned to acquire a full spectrum or set at a
particular mass range window to allow a specific analyte of interest to
pass. Thus, limits of identification for qualitative analysis (e.g., full
spectrum acquisition) and limits of detection for quantitative
determinations (e.g., SIM or MRM) can be improved by removal of
background ions and thereby, e.g., increasing the signal to noise ratio.

[0057]Various embodiments of the present teachings can be used to reduce
noise in mass spectrometric techniques which employ parent-daughter ion
transition monitoring (PDITM), such as for example, SIM or MRM. In
various embodiments, PDITM can be performed on a mass analyzer system
comprising a first mass separator, and ion fragmentor (e.g., a collision
cell) and a second mass separator. The transmitted parent ion m/z range
of a PDITM scan (selected by the first mass separator) is selected to
include a m/z value of one or more of the isobarically labeled
amine-containing compounds and the transmitted daughter ion m/z range of
a PDITM scan (selected by the second mass separator) is selected to
include a m/z value one or more of the reporter ions corresponding to the
transmitted amine-containing compound.

[0058]In various embodiments, the present teachings can provide a means of
reducing the amount of unwanted ions entering an ion trap analyzer and
thus, e.g., reduce space charge effects and increase the dynamic range of
such a mass analyzer. Although using a scanning device in front of an ion
trap can lead to a loss of duty cycle of the trap, rapid scanning and
storage of the ions after the post-reaction band pass filtering of the
ion population could help reduce these losses.

[0059]In various embodiments, the present teachings can be used to reduce
chemical noise in mass spectrometry systems comprising a MALDI ion
source. MALDI spectra, in particular in the low mass region of the
spectra where small molecule molecular ions reside, are often dominated
by chemical noise to a much greater extent than ESI spectra. It is
believed that the majority of this chemical noise is due to matrix
molecules. The problem can be so great as to preclude the use of systems
using MALDI ion sources from qualitative small molecule analytical
applications. In various embodiments, the present teachings can be used
to reduce chemical noise post ionization, yet pre-mass analysis so a
matrixless approach is not required to remove chemical noise. Examples of
MALDI matrix materials for which the methods of the present teaching
might be applied to reduce chemical noise arising therefrom include, but
are not limited to, those listed in Table 1.

[0060]Aspects of the present teachings may be further understood in light
of the following examples, which are not exhaustive and which should not
be construed as limiting the scope of the present teachings in any way.

[0061]All experiments were performed on either a commercial or a custom
modified triple quadrupole mass spectrometers coupled with a HPLC system
(atmospheric pressure ionization, positive mode). The system used in
these examples was an API 365 instrument (MDS Sciex, Inc., Concord,
Ontario, Canada), which is schematically depicted in FIG. 1. The
collision gas inlet was modified to allow for introduction of vapor of a
liquid neutral chemical reagent (e.g., reactive collision gas) into the
collision cell. To perform the noise reduction experiments, the mass
spectrometer was operated in the zero neutral loss (ZNL) scan mode, which
can be used to filter out ions changing m/z values after ion/molecule
reactions with the neutral chemical reagent. Various LC-MS conditions and
types of analytes were tested. The neutral chemical reagent used in these
examples was DMDS.

[0062]The pressure readings noted in the figures and text were obtained
using a Bayet Alpert gauge mounted on the vacuum chamber of the mass
spectrometer, the chamber containing Q1, Q2 and Q3 in FIG. 1. Under
normal Q1 scan operating conditions (no chemical reagent added) the
readout on the gauge was about 6×10-6 torr. When DMDS was
introduced the pressure at the gauge increased to about
1.3×10-5 torr. It should be noted that these pressure readings
have not been corrected for the difference in response of the gauge to
DMDS and nitrogen. Accordingly, the pressure increment (of about
0.7×10-5 torr in this example) is what is referred to as the
"partial pressure" of DMDS. The pressure inside the collision cell was
estimated to be a few millitorr for these operating conditions and
instrument. In principle, without being held to theory, only a single
collision between a neutral chemical reagent molecule and background ion
can be sufficient for reaction to occur.

[0063]Unless otherwise noted, a "partial pressure" of about
0.7×10-5 torr of DMDS (as described above) was used in the
data of this example where DMDS was added.

[0064]FIGS. 4A-17 present examples of the data obtained. A further
understanding of the data in these figures can be had from consultation
of the text and notations made thereon and the brief descriptions
previously presented. FIGS. 18-20 provide a summary in tabular form of
the reactivity of the chemical regent with various analytes and
compounds.

[0065]FIGS. 4A-4B present electrospray mass spectra of the chemical
background spraying of ACN/H2O/TFA in the approximate ratio of
50:50:0.1. FIG. 4A depicting mass spectra without DMDS reaction gas and
FIG. 4B mass spectra with the addition of DMDS to the collision cell. The
reactions occur with an estimated 95% of the total chemical background
ions from this LC/MS mobile phase and others tested with electrospray
ionization. The results indicate that a partial pressure readout on the
Bayart Alpert gauge mounted on the vacuum chamber of the mass
spectrometer of about 0.7×10-5 torr of DMDS, which corresponds
to about 3×10-3 torr in the collision cell of this instrument,
can induce at least one step of reactions between the chemical background
ions and the DMDS.

[0066]FIG. 5 depicts the effect on the total ion current (TIC) when DMDS
was applied and ZNL scanning. FIGS. 6A and 6B depict, respectively, mass
spectra under the conditions of regions (a) and (c) of FIG. 5. The ions
were generated with an electrospray of ACN:iso-propanol:HCOOH. The
regions in FIG. 5 correspond to the following: (a) DMDS added to cell at
a "partial pressure" (as described above) of about 0.7×10-5
torr; (b) no gas added to cell with a background pressure at the gauge of
0.6×10-5 torr; and (c) only nitrogen added to the cell, with a
pressure on the gauge of 0.7×10-5 torr.

[0067]About a 10× reduction in the TIC is observed in this case
attributed to the DMDS and not to additional declustering afforded by the
nitrogen. The TIC remained almost the same between conditions (b) and (c)
in FIG. 5 which indicates the reduction in (a) of chemical background is
due to DMDS. Similar effects have been observed for a variety of other
commonly used LC mobile phases.

[0068]The data of FIGS. 7A-17 were acquired in the zero neutral loss (ZNL)
mode. Data noted as without DMDS, were acquired with nitrogen in the
collision cell, and data noted as with DMDS were acquired with DMDS in
the collision cell. Data presented, showing the reaction products and/or
the extent of reaction of DMDS with the various compounds tested, were
obtained by acquiring a product ion spectrum of the molecular ion of
interest with DMDS in the cell, at very low collision energy (e.g., 2
eV), and scanning above and below the mass of the parent ion.

Prazepam

[0069]FIGS. 7A-7B and 8, present data on prazepam (C19H17ClN2O; MW 324.1)
a high proton affinity compound whose structure is schematically
illustrated as an inset in FIGS. 7A and 8. FIG. 7A presents a Prazepam
ZNL MS spectra without DMDS and FIG. 7B with DMDS added as a neutral
chemical reagent for chemical noise reduction. FIG. 8 mass spectral data
used to ascertain the extent of reaction of DMDS with prazepam
([M+H].sup.+); using a product ion scan of m/z 325 scanning Q3 from about
200 m/z to about 500 m/z with DMDS in collision cell. The reactivity of
DMDs with prazepam was observed to be less than about 1%.

Midazolam

[0070]FIGS. 9A-9C present data on midazolam (C18H13ClFN3; MW 325) a high
proton affinity compound whose structure is schematically illustrated as
an inset in FIG. 9A. FIG. 9A presents a midazolam ZNL MS spectra without
DMDS and FIG. 9B with DMDS added as a neutral chemical reagent for
chemical noise reduction. FIG. 9c (inset in 9B plot) shows mass spectral
data used to ascertain the extent of reaction of DMDS with midazolam
([M+H].sup.+); using a product ion scan of m/z 325 scanning Q3 from about
200 m/z to about 500 m/z with DMDS in collision cell. No reaction
products were observed.

[0072]FIGS. 10A-10B compare ZNL mass spectra of fludrocortisone without
DMDS (FIG. 10A) and with (FIG. 10B) and added to the collision cell.
Background is reduced and the molecular ion remains substantially
unattenuated. The sodium adduct [M+Na].sup.+, at m/z=403, is observed to
be reduced relative to the protonated fludrocortisone [M+H].sup.+.

[0073]FIGS. 11A-11B assess reactions of fludrocortisone with DMDS using
the product ion scan method, Figure. Two thirds of the [M+Na].sup.+ ion
(m/z about 403) were observed to react with the reagent DMDS (producing
peak at about m/z 497, circled by a dashed line in FIG. 11A) (see data of
FIG. 11A). The protonated fludrocortisone ion [M+H].sup.+ (m/z about 381)
showed less than 5% reactivity (reaction product about m/z 475 circled by
a dashed line in FIG. 11B) (see data of FIG. 11B).

Estrone

[0074]FIGS. 12A-12B compare ZNL mass spectra of estrone (C18H22O2, MW
270.4), a relatively low proton affinity compound, whose structure is
schematically illustrated as an inset in FIG. 12B. FIG. 12A presents data
without DMDS and FIG. 12B with DMDS added to the collision cell. The
ammonium adduct of estrone [M+NH4].sup.+ (m/z about 288) shows
approximately a 30% attenuation, while the sodium adduct [M+Na].sup.+
(m/z about 293) was reduced significantly. The background reduction was
also extensive. It was also observed that protonated estrone [M+H].sup.+
(m/z about 271) and the ammonium adduct do not loose substantial ion
current but that the sodium adduct does loose substantial ion current
upon addition of DMDS.

Flunitrazepam

[0075]FIGS. 13A-13B assess reactions of protonated and sodiated
flunitrazepam (C16H12FN3O3, MW 313) with DMDS using product ion scanning.
The chemical structure of flunitrazepam is schematically depicted by the
inset in FIG. 13A.

[0076]Protonated flunitrazepam [M+H].sup.+ (m/z about 314) was observed to
substantially not react to form products with DMDS (m/z about 408) (see
data of FIG. 13A). The sodium adduct, [M+Na].sup.+ (m/z about 336) was
observed to react to a similar extent (reaction product at about m/z 430
and circled by a dashed line in FIG. 13B) as observed for fludrocortisone
(see data of FIG. 13B).

Etamivan

[0077]FIGS. 14A-14B assess reactions of protonated and sodiated etamivan
(MW 223.3) with DMDS using product ion scanning. The chemical structure
of etamivan is schematically depicted by the inset in FIG. 14A.

[0078]Protonated etamivan [M+H].sup.+ (m/z about 224) was observed to
substantially not react to form products with DMDS (see data of FIG.
14A). The sodium adduct, [M+Na].sup.+ (m/z about 246) was observed to
react to a similar extent (reaction product at about m/z 340 and circled
by a dashed line in FIG. 14B) as observed for fludrocortisone and
flunitrazepam (see data of FIG. 14B).

Cyclosporine A

[0079]FIGS. 15A-15B compare ZNL mass spectra of cyclosporine A (MW
1202.6), a relatively low proton affinity peptide (no basic residues)
without DMDS (FIG. 15A) and with DMDS (FIG. 15B) and added to the
collision cell. The chemical structure of is cyclosporine A schematically
depicted by the inset in FIG. 15B. The double protonated cyclosporine ion
[M+2H]2+, at about m/z=602, appears to have gained signal in the
presence of DMDS. The satellite ions (1502) to the doubly charged ion are
the Na and K adducts. The Na adduct is reduced relative to the other
molecular ions to a greater extent by the DMDS but the effect does not to
be as great as with the previous small molecule examples.

Angiotensin II

[0080]FIGS. 16A-16D and 17 present data for angiotensin II. The chemical
structure of angiotensin II is schematically depicted by the inset in
FIG. 17.

[0081]FIGS. 16A-16D compare angiotensin II background reduction with DMDS
under various conditions, where the angiotensin II was ionized by ESI
from a mobile phase of methanol:water:acetic acid in the approximate
ratio of 50:50:0.1.

[0082]FIGS. 16A (a) and 16B (b) compare a Q3 single MS scan (with N2 in
the collision cell) with a zero neutral loss with nitrogen. This
comparison shows that the ion current is reduced by about 2.5-3× by
virtue of transmission losses to be expected when operating two RF/DC
quadrupoles instead of one. Mainly background ions were observed for the
conditions of FIG. 16A. FIG. 16C (c) shows the effect of DMDS at a
partial pressure (as described above) of about 0.7×10-5 torr.
No signal attenuation of the double protonated analyte [M+2H].sup.+ is
observed (compare to 16B (b)) while background reduction is observed to
occur. Fragment ions (e.g., y2.sup.+, a5.sup.+, a6.sup.+,
b5.sup.+ and b6.sup.+) were seen in both cases (b) & (c). A
measurement at a higher DMDS partial pressure (as described above) of
about 1.0×10-5 torr was not observed to improve the spectra
and attenuate the signal by about a factor of 4.

[0083]FIG. 17 depicts a product ion scan of the [M+2H]2+ ion of
angiotensin II with DMDS in the collision cell and a 2 eV collision
energy. No reaction of angiotensin II with DMDS was observed.

Further Data

[0084]FIGS. 18-20, present, respectively, tables with data on other
molecules tested. Chemical structures of various compounds listed in the
tables of FIGS. 18-20 are presented in FIGS. 30-38. Tables 18-20
summarize the reactivity to DMDS of 41 compounds with widely varying
chemical properties and functional groups. Ten of these compounds
produced fragments as well as protonated molecular ions and the
reactivity of the fragments is included. The reactivity of the sodium
adducts as well as other unidentified adducts is also presented. Of the
41 species the majority (30) reacted less than 5%. Thirty eight of the 41
reacted less than 20%. Three of the 41 tested compounds "reacted"
substantially (between 20-25% reacted). Only one of these three compounds
reacted by adduction. The other two compounds did not react, but
fragmented via CID channels. The majority of the compounds that produced
sodiated species showed a high reactivity (>65%) toward that adduct.

[0085]In Tables 1-3 (FIGS. 18-20), the second column gives the name of the
compound tested; the third column provides a list of likely reaction site
for reaction with DMDS. The fourth column indicates the approximate m/z
value of the protonated compound and in parenthesis the approximate
percentage of the protonated compound that reacted with DMDS; the fifth
column indicates the approximate m/z value of the sodiated compound
(sodium adduct) and in parenthesis the approximate percentage of the
sodiated compound that reacted with DMDS; the sixth and last column list
the reaction of various other ions where the number is the ion's
approximate m/z value and the number in parenthesis is the approximate
percentage of that ion that reacted with DMDS.

[0086]The underlined numbers represent those losses arising from
dissociation of the ion and not necessarily adduct formation with DMDS.
The superscript to a mass indicates the charge stat of the ion, e.g.,
cyclosporine A was observed in a double charge state (602, where m=1204
and z=2+) and a singly charged state (m/z=1203).

[0087]In the experiments it was observed that major chemical background
ions reacted with the neutral chemical reagent, Dimethyl Disulfide (DMDS,
CH3S--SCH3), to form adduct ions and fragments thereof. The majority of
the tested protonated analytes, such as the tested peptides including
cysteine containing peptides and multiply charged protonated species,
small molecule pharmaceuticals and other biomolecules, did not react
significantly with DMDS to the same extent that DMDS reacted with the
background ions. It was observed that sodiated molecular ions,
[M+Na].sup.+, reacted to a greater degree than protonated [M+H].sup.+ or
[M+NH4].sup.+ ions on all compounds tested in these experiments.

Background Ions

[0088]FIGS. 21A-24B present data obtained on the reaction of the neutral
chemical reagent of these examples, DMDS, with various background ions.
The data were obtained using product ion scans of targeted background ion
species, adding reactive gas to the cell, and scanning above and below
the mass of the parent background ion. The data show that the vast
majority of electrospray background ions from typical LC solvents react
with DMDS.

[0089]FIGS. 21A-21C assess the reactions of the background ion m/z 99 at
different partial pressures (as describe above) of DMDS using product ion
scanning above and below the mass of the targeted ion. This m/z=99 ion
was determined to be P(OH)4.sup.+ and is schematically illustrated,
e.g., in FIG. 26. The data are for the electrospray ionization of the
output from an LC column with a mobile phase of methanol:water:acetic
acid in the approximate ratio of in the approximate ratio of 50:50:0.1.

[0090]FIG. 21A shows data for a DMDS partial pressures of about
0.4×10-5 torr; FIG. 21B of about 0.7×10-5 torr; and
FIG. 21C of about 1.0×10-5 torr as measured at the Bayet
Alpert gauge as described above. The m/z values for water clusters
[M+nH2O].sup.+, single DMDS adduct water clusters
[M+DMDS+nH2O].sup.+, double DMDS adduct waters clusters
[M+2*DMDS+nH2O].sup.+, triple DMDS adduct waters clusters
[M+3*DMDS+nH2O].sup.+, and DMDS a clusters [M+n*DMDS].sup.+, are
indicated in the figure for ease of evaluation.

[0091]FIGS. 22A-22D assess the reactions of four background ions as
indicated in the figure header, m/z=83, m/z=115, m/z=143, and m/z=159,
respectively. The data are for the electrospray ionization of the output
from an LC column with a mobile phase of methanol:water:acetic acid in
the approximate ratio of in the approximate ratio of 50:50:0.1; and a
partial pressure of about 0.7×10-5 torr of DMS was used as
described above.

[0092]The reactions were observed to be dominated by the formation of DMDS
adducts [M+n*DMDS].sup.+ with up to three neutral DMDS molecules,
combined with addition of water molecules, e.g., [M+nH2O].sup.+.
Water can arise as an impurity in the DMDS and/or as present in the
vacuum background. Various reactions of these ions are illustrated in
FIGS. 25 and 26.

[0093]FIGS. 23A-F assess the reactions of an additional six background
ions which did not show extensive adduction but proceed by charge
transfer. The spectra of FIGS. 23A-F are, respectively, the product ion
scans of (a) m/z 149; (b) m/z 60; (c) m/z 78; (d) m/z 83; (e) m/z 99; and
(f) m/z 205. The data are for the electrospray ionization of the output
from an LC column with a mobile phase of methanol:water:acetic acid in
the approximate ratio of in the approximate ratio of 50:50:0.1; and a
partial pressure of about 0.7×10-5 torr of DMS was used as
described above.

[0094]A charge exchange reaction of the DMDS adduct with the background
ion is observed to occur resulting in m/z 141=[DMDS+SCH3].sup.+. It
is believed to arise by the adduction of several DMDS molecules to the
ion followed by charge exchange to and fragmentation of the DMDS dimer.
This can be an important mechanism to remove phatlates (m/z=83, 149 and
205 in this example). For example, m/z=149 corresponds to a phthalate
background ion that is ubiquitous in most electrospray spectra. The
conversion of 149 to 141 in the spectrum can be used, for example, to set
a minimum band width of a post-reaction bandpass mass filter. In the
examples, the bandpass width was 1 amu for both pre and post reaction
region filters when the mass spectrometer system was used in zero neutral
loss (ZNL) mode.

[0095]FIGS. 24A-B schematically depicts a summary of the reactivity and
believed reaction channels of the background ions in a typical ESI
spectrum of this example from a LC column with a mobile phase of
methanol:water:acetic acid in the approximate ratio of in the approximate
ratio of 50:50:0.1; and a partial pressure of about 0.7×10-5
torr of DMS as described above.

[0096]A few of the background ions showed substantially no reactivity
(circled ions). A legend describing the various reactions leading to
various observed peaks is inset below the spectra, where the solid line
indicates addition of neutral DMDS, the dotted line addition of water,
the diamond-headed line the addition of SCH3 or HSCH3 and the
circle indicating ions that showed substantially no reactivity with DMDS.
FIG. 24B obtained by neutral gain scan (DMDS present in the cell) shows
the chemical background ions that react with at least one DMDS to gain a
mass of 94.

[0097]The identity of many of the background ions has also been elucidated
by MS/MS. FIGS. 25 and 26 schematically summarize a study undertaken to
identify common background ions using various MS/MS scan modes and to
establish the relationships among the background ion populations. The
results are presented as possible "family trees" of chemical background
ions commonly observed from API-LC/MS mobile phases ACN/H2O/HCOOH and
MeOH/H2O/CH3COOH.

[0098]The numbers in FIGS. 25 and 26 refer to the m/z value of a singly
charged ion. The results obtained in these experiments indicate that the
majority of the major chemical background ions are either stable ions (or
fragments thereof) of contaminants, such as adipates, sebacates,
phthalates, phenyl phosphates, silicones and their derivatives (e.g.,
airborne, from the tubing and/or mobile phases, etc) as shown, e.g., in
FIG. 25; or cluster related ions (e.g., solvent/buffer involved) as
shown, e.g., in FIG. 26. The cluster related ions mostly have some ions
from contaminants as nuclei. The neutral molecules of water, methanol,
acetonitrile, and acetic acid are found to be involved in clustering.
Although the intensity and/or appearance of some background ions can vary
under different LC/MS experimental conditions, most observed
cluster-related background ions in these experiments were relatively
stable and survived the declustering conditions in the ion source and
entrance optics.

Mixtures of Analytes

[0099]FIGS. 27A-29D present chromatographic data and data on mixtures of
analytes of interest. The data were obtained using a TurboIon Spray
source off the LC/MS.

[0100]FIGS. 27A-27F depict TurboIon Spray LC/MS mass spectra of four
pharmaceutical compounds, at 200 μL/min, nicotinamide, etamivan,
flunitrazepam, and testosterone, without DMDS (FIGS. 27A, 27B and 27C)
and with DMDS (FIGS. 27D, 27E and 27F). In FIGS. 27A-27F, neutral loss
scanning was performed for the background reduction acquisition and Q3
single MS scans were performed for the standard acquisition. Under these
operating conditions, approximately a factor of 2-3 loss in signal is
expected due to transmission differences so, for comparison purposes, the
TIC baseline is overestimated for the non-background reduced chromatogram
as is the analyte signal in the spectra.

[0101]FIG. 27A shows a base-peak chromatogram (Q3 scan) before addition of
DMDS, and FIG. 27D after. It can bee seen that in the ZNL scan after the
introduction of DMDS (FIG. 27D) the substantial reduction in background
and baseline noise (compare for example portions circled by a dashed
line) and the observation of nicotinamide and testosterone.

[0102]FIGS. 27B and 27E compare the noise reduction in chemical background
mass spectra of the TIC region between about 3 to about 8 min, a region
anticipated to contain some common contaminants, e.g., phthalates such as
m/z=149. The reduction in chemical background noise is clear.

[0103]FIGS. 27C and 27F compare the noise reduction of the TIC region at
about 17.38 min (the approximate retention time of testosterone in this
experiment). The in crease in signal-to-noise testosterone (m/z about
289) after introduction of DMDS is clear as well as a change in the mass
spectra. The signal level in the background reduced testosterone spectrum
FIG. 27F (7000 cps) was observed to be approximately 3× lower than
in the non-background reduced spectrum FIG. 27C (25,000 cps). What is not
accounted for by transmission loss (2-3×) and reactivity of
testosterone with DMDS (a reactivity of about 8% was expected, see e.g.,
FIG. 18) is believed to be due to the removal of isobaric interferences
from the background ions.

[0104]FIGS. 27A-27F provide an example of a practical application of the
neutral chemical reagent DMDS for the reduction of chemical background
noise in LC-MS. FIGS. 27A-27F can be used as an example of the ability of
various embodiments of the present teaching to be used in providing base
peak chromatograms. Base peak chromatograms are often used to reveal the
trace components in LC-MS analysis to localize/identify unknown species.
This approach can be used, e.g., to reduce or prevent the significant
contribution of chemical background ions to a TIC, which can, e.g.,
totally overshadow the appearance of those low abundant analytes. In an
automatic identification or screening process with LC-MS it can be
important to trigger a tandem MS/MS scan to acquire further information
on structures. Such scans are often triggered to perform MS/MS
experiments on the base peak or the most intense ones. However, if the
intensities of the trace components are already lower than that of the
major (base peak) chemical background ions in a mass spectrum, these
minor analytes will not be identified and picked up for a further MS/MS
experiment.

[0105]FIGS. 27A-27F show that, after the chemical noise reduction with
DMDS according to the present teachings, the two minor components
nicotinamide (at about the retention time 2.23 min., i.e., 2 minutes, 14
seconds) and testosterone (17.47 min., i.e., 17 minutes, 28 seconds) are
detected, see, e.g., FIG. 27D, in contrast to the analysis without the
chemical noise reduction, see e.g., FIG. 27A. The signal-to-noise ratio
of the peaks in the base peak chromatogram improves by about a factor of
10-20. The fluctuating baseline before the noise reduction (circled
portion on the right hand side of FIG. 27A) becomes a relatively flat
line after the noise reduction (circle portion on the right hand side of
FIG. 27D). The change of the mass spectra of the component testosterone
before and after the DMDS noise reduction (FIGS. 27C and 27F
respectively) illustrates that background ions have been removed from the
TIC.

[0107]The data before addition of DMDS is a Q1 full scan acquisition and
the data after DMDS addition is a zero neutral loss (ZNL) scan. A
2-3× reduction in transmission efficiency is expected and partially
accounts for the difference in counts on the molecular ions; removal of
isobaric interferences is a possibility as well.

[0108]FIGS. 28A and 28B show that before noise reduction using the present
teachings, only two of the eight biomolecules are detected (FIG. 28A),
but that after (FIG. 28B) all eight are observed. FIGS. 28C and 28D
compare the spectra observed for the TIC region around 10:20, elution of
etamivan. The addition of DMDS according to the present teachings, can be
seen to have increased the protonated etamivan signal (m/z about 224) and
decreased the relative proportion of fragmentation (e.g., peak at about
m/z 149 in FIG. 28c and peak at about m/z 151 in FIG. 28D).

[0109]FIGS. 29A-29D depict TurboIon Spray LC/MS chromatograms (FIGS. 29A,
29B) and mass spectra (FIGS. 29C, 29D) of a mixture of five biomolecules:
nicotinamide (RT=2:09), [M+H]+=123; norfloxacin (RT=6:53), [M+H]+=320;
etamivan (RT=10:15), [M+H]+=224; flunitrazepam, (RT=13:10), [M+H]+=314;
and testosterone (RT=14:05), [M+H]+=289, without DMDS (FIGS. 29A and 29C)
and with DMDS (FIGS. 29B and 29D). The mixture comprised about 10 ng of
each biomolecule. The data before addition of DMDS is a Q3 single MS scan
with nitrogen gas and the data after DMDS addition is a zero neutral loss
(ZNL) scan. It is to be understood that in FIGS. 29C and 29D, the loss of
norfloxacin signal (9000->5000 cps) largely due to transmission losses
due to the change in scan mode.

[0110]FIGS. 29A and 29B compare TIC chromatograms and demonstrate the
ability, in various embodiments, of the present teachings to reveal
signals otherwise obscured by noise. For example, by the neutral chemical
reagents of the present inventions reacting with one or more contaminants
but not substantially reacting with one or more analytes of interest. For
example, two trace components (nicotinamide and norfloxacin) at the
retention times of 2.15 and 6.90 min., respectively, where detected in
the basepeak chromatogram after the chemical noise reduction with DMDS
(see FIG. 29B) that were note observed before (se FIG. 29A).

[0111]FIG. 29c (without DMDS) and 29D (with DMDS) compare the spectra
observed for the TIC region around 6.96 min., elution of norfloxacin. The
addition of DMDS according to the present teachings, can be seen to have
increased the protonated norfloxacin signal (m/z about 320) and relative
to the noise.

[0112]FIGS. 30-38 depict chemical structures of various compounds listed
in the tables of FIGS. 18-20. In addition, FIGS. 30-38 summarize some of
the data regarding the reaction of the protonated forms of these
compounds with DMDS. The percentage listed next to structure indicate the
observed reactive percentage of the protonated molecule. Underlined
percentages indicate the reactions are dissociations. In some instances,
analogs derived from a compound in the list were also studied and their
reaction percentage are also indicated, e.g., such as loss of water from
a hydrated analog.

[0113]All literature and similar material cited in this application,
including, but not limited to, patents, patent applications, articles,
books, treatises, and web pages, regardless of the format of such
literature and similar materials, are expressly incorporated by reference
in their entirety. In the event that one or more of the incorporated
literature and similar materials differs from or contradicts this
application, including but not limited to defined terms, term usage,
described techniques, or the like, this application controls.

[0114]The section headings used herein are for organizational purposes
only and are not to be construed as limiting the subject matter described
in any way.

[0115]While the present teachings have been described in conjunction with
various embodiments and examples, it is not intended that the present
teachings be limited to such embodiments or examples. On the contrary,
the present teachings encompass various alternatives, modifications, and
equivalents, as will be appreciated by those of skill in the art.

[0116]While the teachings have been particularly shown and described with
reference to specific illustrative embodiments, it should be understood
that various changes in form and detail may be made without departing
from the spirit and scope of the teachings. Therefore, all embodiments
that come within the scope and spirit of the teachings, and equivalents
thereto, are claimed. The descriptions and diagrams of the methods,
systems, and assays of the present teachings should not be read as
limited to the described order of elements unless stated to that effect.